The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition
Abstract Have you heard that garlic powder is supposed to inhibit the growth of bacteria? Which do you think would make a better disinfectant: a solution of garlic powder or a solution of bleach? This project shows you a straightforward way to compare the effectiveness of different disinfectants (or other antimicrobial agents), by measuring zones of inhibition on a culture plate. Objective The goal of this project is to measure the effectiveness of different antimicrobial agents by measuring zones of inhibition on bacterial culture plates. Materials and Equipment Note: If you are carrying out this experiment in a school laboratory, some of the materials and equipment listed below may be more readily accessible. These items can be purchased from Carolina Biological Supply Company, a Science Buddies Approved Supplier: *Sterile disks (3 per disinfectant tested). Alternatively, disks may be made using a hole punch and filter paper, but they will need to be sterilized in the oven, as described in the Procedure. *Nutrient agar plates (6): **3 plates will serve as controls, with no disinfectants, **3 plates will serve as test plates, with disinfectant disks. *Sterile cotton-tipped applicator swabs. Alternatively, cotton-tipped swabs from a new, unopened box may be used. *Live E. coli, strain K-12 *Forceps. Alternatively, tweezers may be used. *70% ethanol or isopropyl rubbing alcohol, for using sterile technique. Alternatively, this can be purchased in the disinfectant section of a grocery store or drug store. Note: If you are ordering this chemical through Carolina Biological Supply Company, the chemical must be ordered by a teacher and shipped to a school or business address, so plan accordingly. You will also need to gather these items *Permanent marker *Optional: Pencil *Optional: Aluminum foil and oven *Timer or clock *Disposable gloves. Can be purchased at a local drug store or pharmacy, or through an online supplier like Carolina Biological Supply Company. If you are allergic to latex, use vinyl or polyethylene gloves. *Disinfectants (up to 6). Here are some ideas for different compounds to test: **Solution of garlic powder **Liquid bathroom cleaner **Liquid floor cleaner (e.g., one containing pine oil) **Mouthwash **Contact lens cleaner **Anti-acne product **Household bleach (sodium hypochlorite) *Optional: 37°C incubator for bacterial culture plates *Ruler, metric *Lab notebook Introduction Antimicrobial agents are chemicals that are used against bacteria. There are many such agents available. Because there are many different situations where bacterial control is important, no antimicrobial agent is effective in all situations. For example, you wouldn't use the same compound to fight an ear infection as you would use to sterilize surfaces in an operating room. The situations are completely different. In one case, you are trying to assist the body to fight off an internal infection, and in the other case, you are trying to eliminate bacteria from inanimate surfaces. There are many additional factors that you would have to consider in order to choose an appropriate antimicrobial agent for a given situation. For example, are the chemical properties of the agent (e.g., pH and solubility) appropriate for the situation? You would want to know whether the compound is toxic—to humans, other animals, plants, or beneficial bacteria. Finally, you would definitely want to know that the compound is effective against the organism(s) you are trying to eliminate. This project shows you one method of measuring the effectiveness of an antimicrobial agent against bacteria grown in culture. This is called the Kirby-Bauer disk-diffusion method, and here is how it works. The bacteria of interest is swabbed uniformly across a culture plate. Then a filter-paper disk, impregnated with the compound to be tested, is placed on the surface of the agar. The compound diffuses out from the filter paper into the agar. The concentration of the compound will be higher next to the disk, and will decrease gradually as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow wherever the concentration in the agar is greater than or equal to that effective concentration. This region is called the "zone of inhibition." Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound. Figure 1, below, illustrates the idea. You can use this method to compare the effectiveness of different disinfectants or different antibiotics against a strain of bacteria. Since this method depends on diffusion of the compound, it is important to keep several factors constant when you make your comparisons, including: *the size of the filter disks, *the temperature of incubation, *the composition and thickness of the agar, and *the uniformity of bacterial plating. With careful attention to making your conditions consistent, this method will produce reliable results for comparing antimicrobial effectiveness. Terms and Concepts To do this project, you should do research that enables you to understand the following terms and concepts: *colony, *zone of inhibition, *disinfectant, *antibiotic, *antiseptic. Experimental Procedure Working with Biological Agents'For health and safety reasons, science fairs regulate what kinds of biological materials can be used in science fair projects. You should check with your science fair's Scientific Review Committee before starting this experiment to make sure your science fair project complies with all local rules. Many science fairs follow Intel® International Science and Engineering Fair (ISEF) regulations. For more information, visit these Science Buddies pages: Projects Involving Potentially Hazardous Biological Agents and Scientific Review Committee. You can also visit the webpage ISEF Rules & Guidelines directly. This science fair project involves the use of the bacteria ''E. coli. While E. coli is not considered a biohazardous or dangerous bacteria, it is important to always properly clean and dispose of bacteria and supplies that come in contact with it. See the Bacterial Safety guidelines below for more details on how to handle bacterial cleanup and waste. Preparing Plates for Disk Diffusion Test For this experiment, it is important to innoculate the plate with a uniform distribution of bacterial colonies, and to use the exact same procedure for each plate. Here are the steps for innoculating the control and test plates. #You can use a pencil or permanent marker to label each sterile disk with a code for the disinfectant to be used for that disk (up to six). If you label the disks, keep track of the codes in your lab notebook, wrap the disks in aluminum foil, and sterilize in a 300° oven for 30 minutes. #Use a permanent marker to mark the bottoms of the three nutrient agar plates that will be your test plates with as many sections as you have disinfectants (up to six). The sections should all be equal in size. Number the sections sequentially. #Label the three nutrient agar plates that will be your control plates. The purpose of these plates is to show that the bacteria consistently grow uniformly over the plate in the absence of disinfectant disks—confirming that your innoculation technique is consistent, and that the plates support uniform bacterial growth. #Using proper sterile technique, inoculate each plate uniformly. Dip a sterile cotton-tipped applicator swab in the K-12 E. coli bacterial solution. Gently wipe the swab over the surface of the plate, swabbing in three directions (120° apart) to insure complete coverage of the plate. Cover the plate and wait at least five minutes for the plate to dry. #Hold a single sterile disk by the edge with sterile forceps and dip it into the disinfectant solution to be tested (make sure it matches with the label on the disk). Touch the disk against the side of the container to drain off excess liquid. #Use sterile forceps to place a single disinfectant disk in the center of each of the marked sections on your test plates. Use the forceps to gently press each disk against the agar surface to insure good contact. Remember to use the exact same technique for each disk—consistency is very important for this experiment. Take notes in your lab notebook to keep track of which disinfectant is tested in each numbered section. #Incubate all of the plates, inverted (agar on top), overnight at 37°C. Use a longer incubation time if necessary (for example, for incubation at lower temperature). Measuring Zones of Inhibition #After overnight incubation, examine your plates (keep them covered at all times). ##The control plates should show uniform colonies over the entire surface of the plate. If the distribution is highly uneven, you will need to improve your innoculation technique and repeat the experiment. ##If your disinfectants are effective at the concentrations you tested, you should see zones of inhibition around the disinfectant disks. The clear zones around each disk should have a uniform width, since diffusion of the compounds through the agar should be uniform in every direction. If this is not the case, suspect either your impregnation technique, or poor contact of the filter paper with the agar. #Measure the diameter of the zone of inhibition for each disk. Keeping the lid of the plate in place, use a ruler to measure the diameter of the disk plus the surrounding clear area in millimeters (mm). ##Include the diameter of the disk in your measurements. For example, if your disk has a diameter of 6 mm and the clear area has a width of 3 mm beyond the disk, the diameter of the zone of inhibition that you should measure and record would be 12 mm (6 mm + 3 mm + 3 mm). This is the standard way that zones of inhibition are measured. ##You will get three separate measurements for each disinfectant, one from each of the three test plates. #Are the diameters consistent across all three plates? Calculate the average and the standard deviation of the diameter of the zone of inhibition for each disinfectant. #Use the values from Table 1 (below) to evaluate the bacterial response to each compound (Johnson and Case, 1995). Bacterial Safety Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all bacterial cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders. You can also see the Microorganisms Safety Guide for more details. Additionally, many science fairs follow ISEF Rules & Guidelines, which have specific guidelines on how bacteria and other microorganisms should be handled and disposed of. *Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria. *Make sure to wash your hands thoroughly after handling bacteria. *'''Proper Disposal of Bacterial Cultures **Bacterial cultures, plates, and disposables that are used to manipulate the bacteria should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours. **Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes. **After bleach treatment is completed, these items can be placed in your normal household garbage. *'Cleaning Your Work Area' **At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used. **Be aware of the possible hazards of disinfectants and use them carefully. Photos Category:Microbiology Experiments